PROJECT SUMMARY: Reprogramming somatic cells to become pluripotent stem cells holds great promise for stem-cell-based therapeutics, but one of the major barriers lies in the ability to identify which of the stem cells are truly pluripotent. We have shown that induced pluripotent stem cells (iPSCs) from mice can have markedly different potential to generate ?all-iPS? animals, even when the cells have similar transcription profiles. This difference suggests an essential role for epigenetic mechanisms in regulating stem cell pluripotency. When developmental bivalent (DB) genes are activated in lineage commitment, they are silenced but poised for later activation in pluripotent stem cells. We currently have no way to probe for this necessary poised state, as transcriptional profiles of iPSCs do not reveal potential for transcriptional activation. We previously reported that H3.3 is required to establish pluripotency during reprogramming and is required to maintain the bivalency of DB genes in ESCs. In fact, our preliminary data showed that H3.3 is enriched at the promoter of many DB genes, and its lack of enrichment correlates tightly with compromised developmental potential in iPSCs. This finding indicates that H3.3 plays a critical role in regulating DB gene expression, and thus pluripotency. Based on our observations, we hypothesize that H3.3 is required to establish bivalency during reprogramming and that this epigenetic signature at the promoter poises DB genes for later activation upon differentiation. Our long-term goal is to elucidate the key mechanisms of establishing and maintaining pluripotency in stem cells. The objective of this proposal is to define the mechanisms by which the histone variant H3.3 regulates the DB genes and the developmental properties of stem cells. We plan to test the hypothesis using unique animal models that will permit us to obtain enough genetically uniform cells at any intermediate stage. This will allow us to study both H3.3 and histone modifications using ChIP sequencing during reprogramming. We propose the following two aims in this application: Aim 1: Identify how H3.3 regulates the establishment of epigenetic signatures in DB genes during reprogramming toward pluripotency. Aim 2: Determine the function of H3.3 enrichment mark at the promoter in DB genes during iPSC differentiation. Successful completion of these aims will allow us to identify how the enrichment of H3.3 at the promoter for DB genes impacts the potential for differentiation of stem cells into specific cell lineages. This work will provide both key information on the functional relevance of epigenetic regulation of pluripotency, and also a possible clinical approach to evaluate the pluripotency of stem cells.